DESCRIPTION: A universal single-dose vaccine against influenza remains elusive. Three recent discoveries by the PI's research team show that the delivery of influenza subunit antigens by recombinant outer membrane vesicles (rOMVs) derived from the Nissle E. coli strain (EcN) may lead to an effective single-dose vaccine against influenza. The objective is to investigate each of these discoveries in greater depth to gain a greater understanding of their vaccine potential. The hypothesis is that a safe and effective single-dose universal vaccine against influenza will result from the controlled release of rationally designed EcN-derived rOMVs decorated with influenza antigens. The hypothesis will be evaluated in the following Specific Aims: Specific Aim 1: To determine the cross reactivity and heterosubtypic immunoprotection profiles conferred by hemagglutinin (HA) variants delivered by EcN-derived rOMVs. HA subunits from known pandemic/epidemic strains of influenza will be expressed in EcN-derived OMVs and subcutaneously administered to BALB/c mice. Humoral and cellular responses will be quantified by ELISA and T-cell proliferation/cytokine expression. Heterosubtypic immunoprotection will be determined by hemagglutination inhibition, infection blockage and infection challenge in BALB/c mice. The anticipated outcome is an in-depth understanding of the heterosubtypic immune response and protection potential of an EcN-derived rOMV vaccine against the most prevalent pandemic and epidemic influenza strains. Specific Aim 2: To determine the humoral and cellular response to the conserved extracellular domain of the influenza matrix 2 protein (M2e) delivered by EcN-derived rOMVs. Tandem M2e genetic constructs will be expressed in EcN-derived rOMVs and subcutaneously administered to BALB/c mice. Humoral and cellular responses will be quantified by ELISA and T-cell proliferation/cytokine expression. Efficacy of rOMV-delivered M2e will be evaluated by infection blockage and infection challenge in BALB/c mice. The anticipated outcome is an in-depth understanding of the potential efficacy of EcN-derived rOMVs containing M2e as a component of a universal vaccine against influenza. Specific Aim 3: To determine the cross reactivity and heterosubtypic immunoprotection profiles conferred by HA variants delivered by EcN-derived rOMVs as a single-dose formulation. HA-1 will be expressed in EcN-derived rOMVs, formulated into controlled release microspheres and subcutaneously administered to BALB/c mice. Humoral and cellular responses will be quantified by ELISA and T-cell proliferation/cytokine expression. Heterosubtypic immunoprotection will be determined by hemagglutination inhibition, infection blockage and infection challenge in BALB/c mice. The anticipated outcome of Specific Aim 3 is the in-depth evaluation of controlled release rOMV vaccines as a single-dose formulation. The collective outcome will be an in-depth understanding of a translatable approach for influenza vaccination.